Synaptodendritic degeneration correlates with cognitive decline in patients with HIV-1-associated neurocognitive disorders (HAND). A confocal imaging-based assay was developed to detect intact postsynaptic densities (PSDs) based on detection of clusters of the scaffolding protein PSD95 fused to green fluorescent protein (PSD95-GFP) and it was established that fluorescent puncta represent functional synapses. PSD95-GFP puncta were lost following exposure to factors released by HIV-1 infected cells including the HIV-1 protein Tat. The signaling pathways that control synapse number changed during exposure to Tat; one pathway regulated synapse loss and the other regulated synapse recovery. Drugs that reverse excitatory synapse loss are hypothesized to have the greatest potential for inducing cognitive recovery. This application describes the development of a high content analysis (HCA) assay based on synaptic imaging and its use to screen a library of drug-like molecules with the long-term objective to discover drugs that reverse cognitive decline in HAND. This unbiased screen will identify compounds acting via currently unidentified signaling pathways that control synapse recovery during a neurotoxic challenge. In the R21 Phase, a confocal microscope-based synapse recovery assay will be adapted to a HCA platform. Progress will be marked by three milestones: 1) reliable production of cortical neurons expressing PSD95-GFP at sufficient scale and density for high-throughput screening, 2) high resolution imaging of PSD95-GFP and quantitative analysis of puncta counts using the HCA platform, and 3) reproducible and statistically significant synapse recovery produced by positive relative to negative control drugs using the HCA platform. Upon meeting these milestones the HCA assay will be scaled up and automated in the R33 Phase. The automated assay will be validated by screening the library of pharmacologically active compounds (LOPAC). LOPAC results will be used to determine assay window, variance and reproducibility. The HCA will then be used to screen a collection of 10,000 compounds selected from targeted libraries of pharmacologically active and structurally diverse compounds. Cheminformatics will be used to cherry-pick compounds for confirmation and validation. Confirmed compounds will be triaged for non-desired activity such as non-specific synaptogenic effects. At the completion of the screen, the assay will be ready for high throughput-scale implementation and/or lead compound optimization. This project will provide a foundation to guide the development of drugs to improve function in HAND patients and will provide an efficient platform for studies of synaptic function. Because synapse loss is common to many neurodegenerative processes, compounds that test positive in this assay may prove useful as drugs to induce recovery in patients with neurological impairments resulting from multiple etiologies.